The long-range goal of this project is to elucidate the homeoprotein Nkx2.5-dependent regulatory pathways in postnatal cardiomyocytes. Particularly we are focusing on direct involvement of this factor in controlling expression of downstream target genes as a prerequisite to the onset of cardiac disease. In the mouse, Nkx2.5 is expressed throughout heart development from precardiac mesoderm cells to mature adult cardiomyocytes. Mutations in human NKX2.5 cause progressive conduction defects, various cardiac anomalies and occasional left ventricular dysfunction suggesting that this transcription factor plays important roles in formation of the developing heart as well as in maintenance of normal cardiac function after birth. In addition, Nkx2.5 upregulation in acquired heart diseases including cardiac hypertrophy has been addressed. These studies indicate that misregulated expression of Nkx2.5 leads to various sequential pathogenic events. Our hypothesis is that Nkx2.5 actively regulates a critical set of genes in postnatal cardiomyocytes to maintain proper cardiac function. This is supported by preliminary data demonstrating that neonatal Nkx2.5-knockdown cardiomyocytes appear to have altered cytoskeletal organization, and adult Nkx2.5- knockdown cardiomyocytes show reduced contraction and Ca2+ handling. We propose to define Nkx2.5- dependent regulatory pathways at a molecular level in two focused areas, cardiac contraction and conduction, at two different cardiac developmental stages, neonatal (Aim 1) and adult (Aim 2). In addition, these effects observed in a single cardiomyocyte will be directly tested in in vivo whole hearts by transcoronary RNAi-virus delivery (Aim 2). Finally, we will elucidate the involvement of Nkx2.5 in the transcriptional regulation of target genes in vivo through the specific Nkx2.5-DNA binding sequence(s) and the role of Nkx2.5 in the formation of transcription complexes at promoters (Aim 3). Three Specific Aims are proposed: Specific Aim 1: Identify biological roles of Nkx2.5 in neonatal cardiomyocytes using RNAi-mediated gene knockdown. Specific Aim 2: Identify biological roles of Nkx2.5 in adult cardiomyocytes as well as in in vivo heart by transcoronary gene transfer of adenoviral RNAi. Specific Aim 3: Determine molecular mechanisms of Nkx2.5-dependent gene regulation of the GO/G1 switch gene 2, GOS2.